Antiseptic, antiseborrheic and exfoliating composition to remove or prevent acne

ABSTRACT

The invention refers to an antimicrobial, exfoliating and seboregulating hydrosoluble composition comprising a combination of diallyl disulphide modified oxide (DDMO) and alpha hydroxyethanoic acid (glycolic acid), which acts removing bacteria affecting the skin including those caused by acne, said composition also provides deep cleansing, exfoliating action, reduces epidermal cohesion facilitating cellular change, preventing the occurrence of several forms of acne in the skin and additionally producing seboregulating effects by avoiding blockage of sebaceous glands.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/008,210, filed on Jun. 14, 2018, which is a continuation of U.S.application Ser. No. 14/421,616, filed on Feb. 13, 2015, which is a 35U.S.C. 371 National Stage Filing of International Application No.PCT/MX2013/000099, filed on Aug. 22, 2013, which claims priority toMexican Application No. MX/A/2012/009806, filed on Aug. 23, 2012. Thecontents of the aforementioned applications are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a hydrosoluble pharmaceuticalantiseptic, antiseborrheic and exfoliating composition comprising acombination of diallyl disulphide modified oxide (DDMO) and alphahydroxyethanoic acid (glycolic acid) to remove bacteria affecting theskin, besides said composition provides a deep cleansing because itreduces the epidermal cohesion and facilitates the cellular replacement,preventing or removing the occurrence of several forms of alterations inthe skin.

BACKGROUND OF THE INVENTION

The skin has a microbial flora, which can be affected by changes innormal conditions of the skin, more likely acne is the most frequency ofthese alterations, due to the multiple factors altering it and producinga balance loss of the microbial flora.

Acne is an inflammatory illness of the pilosebaceous follicles,associated to the keratinization and seborrhea alterations;characterized by the formation of skin wounds such as: blackheads,papulae, pustules, cysts or abscesses, which frequently leave residualscars.

Acne is undoubtedly the most frequent skin disease in the entire world;traditionally the three main factors considered to intervene in theappearance of acne were microbial infection, overproduction of fat andabnormal peeling of epidermal cells that finally cause an excess in thekeratinization, but recent studies add as causing effects of acnegenetic predisposition and inflammatory factor.

It is calculated that about 85% of the population between the ages of 11and 30 suffer from acne; this represents only in Mexico an approximatetotal of 20 million people. The incidence curves show a maximum peak at18 years old, from this point a gradual decrease is appreciated, whichincreases after 30 years of age; however, between 25 and 35% of adultsover 35 years old go through occasional acne exacerbations.

The cause of acne is precisely unknown, due to the fact that a greatamount of factors jointly intervene, however, among them it is worthmentioning the genetic factor, which in combination with the endocrinal,inflammatory and infectious factors, are somehow the cause of thepolymorphic characteristic of acne.

There are 5 main primary pathogenic factors interacting in a complexmanner to produce acne injuries.

-   -   1. Genetic predisposition.    -   2. Overproduction of fat by sebaceous glands.    -   3. Alteration in the keratinization process with abnormal        peeling of the sebaceous follicular epithelium (causing        comedogenesis).    -   4. Proliferation of Propionibacterium acnes.    -   5. Release of inflammatory intermediaries on the skin, mainly        alpha-TNF and alpha IL-1.

The genetic predisposition is not mediated by one simple Mendeliancharacter, but it is caused by an intermediary of a polygenic mechanism,which originates special receptiveness of the pilosebaceous follicle, inorder to more intensely responds to androgens, different from howhealthy people respond.

Research made about the role of fat in acne has allowed to establishingthat the sebaceous lipids are regulated by the peroxisome proliferatoractivated-receptors (PPAR) and by sterol transcription factors.

Other research of the sebaceous gland function has contributed toobtaining information about the main role these glands play inregulating skin functions. The sebaceous gland has direct and indirectantibacterial activities. Likewise, the sebaceous gland producesantibacterial peptides and proinflammatory cytokines that are induced insebocytes due to the presence of bacteria.

Androgens are hormones synthesized in testicles, ovaries and suprarenalcortex. During puberty, through little known mechanisms, the hypophysisstarts to secrete greater amounts of luteinizing hormones (HL) andfollicle-stimulating hormones (FSH), which together are responsible forthe increase in testicular growth, spermatogenesis, and steroidogenesis.Testosterone acts upon sebaceous glands, increasing their size and fatsynthesis; in women the mechanism is similar. Through biopsies to theface skin in patients with acne, it has been proven that during pubertythe sebaceous glands are bigger and foliated.

Some time ago it was believed that acne was just an infectious processcaused by the bacteria “Acne bacillus”, now called Propionibacteriumacnes. The cutaneous flora is essentially a triad lead by thePropionibacterium acnes, in addition to Staphylococcus epidermis andyeast, which can be Pityrosporum ovale or Pityrosporum orbiculare.

It is possible that the association between bacteria and thepathogenesis of the acne is reinforced by well-documented findings aboutclinical improvement in patients with acne treated with systemicantibiotics. The antibacterial therapy does not affect the P. orbiculareor P. ovale since they reside on the upper part of the acrofundibulum ofthe sebaceous follicle. The anaerobe P. acnes on the other hand seems toplay a central role in the development of acne inflammation. The mostimportant evidence might be the demonstration that therapy withantibiotics results in a significant suppression of P. acnes, which isaccompanied in one reduction in the number of inflammatory injuries.

Propionibacterium acnes and Staphylococcus epidermidis are produces of alipase that hydrolyzes triglycerides from fat in fat-free acids, whichare potent irritants from the follicular channel, when applied on theskin or intradermally injected, they cause inflammation and blackheads.

It must also be noted that the Propionibacterium acnes alternatelyactivates the complement system, this fact has allowed to propose thatthis infectious mechanism could also play an important role in theproduction of acne inflammatory injuries.

During puberty, and in response to the androgens produced in greateramounts in this stage, the sebaceous glands that were relativelyinactive, increase in size and become more foliated, thus increasing theproduction of fat spilled on the outside; thereby the first sign ofacne: seborrhea.

The recently synthesized fat contains triglycerides, squalene and waxesters, and it is known that Propionibacterium acnes through a lipasehydrolyzes triglycerides from this sebaceous material, thus convertingthem into fat free acids, which in addition to other irritant substancesas squalene and oleic acid cause inflammation of the follicular channel,which responds to the inflammation with hyperkeratosis. The resultingcorneous material fall in the light of the follicle, which in additionto the fat excess forms wax substituting and easing the follicle walls,accordingly by not being able to excrete material, it inflames more,causing the first and most important acne elemental injury: theblackhead, causing dilation of the follicular hole due to the pressurethe wax applies when trying to be expelled.

In several studies, it was been studied in depth the role of theinflammatory mediators, as well as the interrelation of this factor withsebaceous lipids and matrix metaloproteinases (MMP's) in the physiologyof acne.

One of the pioneer researches in this field was developed by Jeremy andco-workers in 2003, who researched the initial events of acne injuriesand found that the immunological changes and inflammatory responsesoccurred before the hyperproliferation of keratinocytes, with a similarpattern to a type IV retarded hypersensitivity. An important fact isthat the whole process starts by the exacerbated regulation of IL1-βhaving a proinflammatory action in response to the linoleic acidrelative deficiency caused by the fat excess and by the disturbance ofthe barrier function inside the follicle. Vowels and co-workers provedin an in vitro study the presence of a P. acnes soluble factor that alsoinduces the production of pro-inflammatory cytokines in cellular linesderived from human monocytes. This product of P. acnes induces thesynthesis of the alpha tumor necrosis factor (α-TNF) and interleucine1-beta (IL1-β) in said cellular lines. It was also shown that theinduction of cytokines by P. acnes would happen through the activationof the “TOLL-LIKE-2” Receptor (TLR-2) shooting inflammatory responses.

Other recent studies have proved that this chain of events occurs ininflammatory injuries from patients with facial acne. It has been shownthat the Propionibacterium acnes also induces type Toll receptors. Thisprovides additional evidence that the inflammatory cytokines, which actthrough autocrine and paracrine mechanisms amplifying theircorresponding receptors and amplifying the signaling way that activatesthe Activator Protein-1 (AP-1) that is a transcriptional factor.

In treating acne, the use of antimicrobials is a normal and habitualbehavior among doctors, since there is clinically an improvement inpatients with acne when using this type of products, this due to thestrengthening factor of P. acnes in the fat metabolism that creates anirritant effect causing greater inflammation on the patient.

The diallyl disulphide oxide (DDO) is also known as allicin, is theproduct from the conversion of allina, which is found in garlic (Alliumsativum), by means of allinase enzyme catalysis. It is a sulfuratedcompound containing diverse pharmacological activities of interest. TheDDO is not naturally found in garlic, but when the bulb fractures, it iscut or grinded, liberates allina, which when contacting the allinaseenzyme creates the active principle. Allina is found in amounts varyingfrom 0.22-0.24% by weight of garlic and is an amino acid that is it nota structural part of any protein and it is not biochemically essentialfor human nutrition.

Since it is a not much stable compound, the DDO quickly loses itsproperties, on the other hand if garlic is heated over 60° C. it losesits properties. It has a strong oxidative power that could cause harm tointestinal cells.

It has been shown that DDO has in vitro activity against Candidaalbicans, some Trichomonas, Staphylococcus aureus; Escherichia coli,Salmonella typhi, S. paratyphi, Shigella dysenterica and Vibro choleraspecies.

The structural formula of allicin is shown below:

In 1944 Cavallito and his co-workers were able to isolate and identifythe active compound from the extracts of Allium sativum which theycalled allicin and in 1947 they were able to synthesize the diallyldisulphide oxide (allicin), but unfortunately they were not able to makethe molecule thereof stable, since it kept losing its efficacy in a veryshort period of time. A group of Mexican researchers from 2000 and 2005were able to stabilize the diallyl disulphide oxide, adding a co-factor,compound called diallyl disulphide modified oxide [DDMO], which is muchmore stable than allicin and it seems to maintain its properties.

The diallyl disulphide modified oxide [DDMO], is the chemical compoundcalled [1,2-alpha-glucodiallyl-disulphide oxide, 2-propen+chloride-6alkyl (benzyl, methyl, octyl ammonium-hydroxymethylamine); whosestructural formula is represented as follows:

In diverse studies it has been shown the activity of allicin, howeverits little stability has been a limitation in its possible therapeuticapplications. Accordingly, it is clear that the application of thestability properties of compound DDMO, will allow the therapeuticapplication based on its antibacterial and anti-septic properties amongothers from allicin.

On the other hand, hydroxyethanoic acid (HAA), is also called glycolicacid, which is an acid of small molecular chain having the ability topenetrate the skin faster that deeper layers. It is shown colorless,odorless, and hygroscopic crystalline solid that is highly water-solubleand in related solvents. This acid is largely used in dermatologictreatments, there are studies demonstrating its efficacy, throughmethods such as computerized morphometry on corneal layers frombiopsies. Normally it is employed in association with other dermatologicproducts supporting the cellular reconstruction of the skin, thusbecoming the so-called “soft-peeling” base. The treatment action withhydroxyethanoic acid is to decrease the corneal layer thickness of theskin and to increase the malpighian layer thickness; the fractions ofareas occupied by collagen in dermal layers significantly increases,thereby there is an improvement in the appearance of atrophic scars fromacne and in the treatment of human skin wrinkles. It is also anexcellent exfoliator, thus helping to significantly prevent and combatacne in any body part. The glycolic acid also lets other components tomore easily penetrate the skin, thus it is advisable to also use theso-called complete treatments. The hydroxic acid also has seboregulatingproperties by increasing the peeling of cells and thus avoiding blockageof the sebaceous glands and the formation of blackheads and other acneinjuries.

OBJECT OF THE INVENTION

A first object of the present invention is to provide antimicrobial,exfoliating and seboregulating pharmaceutical hydrosoluble compositionscomprising a combination of diallyl disulphide modified oxide (DDMO) andalpha-hydroxyethanoic acid (glycolic acid) to remove or prevent acne.

A second object is to obtain compositions comprising a combination ofdiallyl disulphide modified oxide (DDMO) and alpha-hydroxyethanoic acid(glycolic acid), antimicrobials/antiseptics, stable, biodegradable andnon-poisonous topical application exfoliants and seboregulators havingas an antimicrobial function a wide action spectrum, not just againstgram negative and gram positive microorganisms, but against fungus aswell.

The previous objects are representative, but they shall not beconsidered as limitative to the present invention, wherein it is furthershown its treatment methods, applications and/or pharmaceutical uses inthe preparation of medicaments to remove, reduce or prevent microbialinjuries of the skin, including acne.

DESCRIPTION OF THE INVENTION

The present invention is related to antimicrobial, exfoliating andseboregulating pharmaceutical hydrosoluble compositions comprising from0.001 to 10% p/w of the DDMO composition, from 1 to 12% p/w ofalpha-hydroxyethanoic acid (glycolic acid), from 0.3 to 24% p/w of oneor more surfactants, from 0.3 to 10% p/w of a moisturizing agent, from0.2 to 10% p/w of an aromatizing agent and from 40 to 70% p/w of thecomposition of an aqueous solvent.

Particularly, the object compositions of the present invention compriseone or more surfactants selected from the group consisting of linealalkyl benzene sulfates and ionic alkyl sulfates.

The surfactants useful in the object compositions of the presentinvention preferably are sodium lauryl sulfate and sodium lauryl ethersulfate.

The ionic alkyl surfactants, such as sodium lauryl sulfate that is ananionic surfactant, are used in a variety of pharmaceuticalnon-parenteral formulations. For example, the sodium lauryl sulfate is adetergent and moisturizing agent, effective in acid, alkaline solutionsand in hard water. It is used in medicated shampoos, such as skincleaner and dentifrices, it is usually known as “Cosmetic Detergent”,makes foam and bubbles and is good for removing grease and oil from skinand hair.

Likewise, the sodium lauryl ether sulfate is the most frequently usedactive anion surfactant in the manufacture of cleaning, scrubbing anddomestic washing products, as well as cosmetic and personal hygieneproducts. The sodium lauryl ether sulfate does not irritate the skin andif from natural origin; additionally it is valued for its easydegradations and properties in relation to the formation of foam. Thesodium lauryl ether sulfate is mainly used in the production process ofshampoos, liquid soaps, face skin creams, dental creams, etc.

The surfactant preferably used in the present invention are sodiumlauryl sulfate, sodium lauryl ether sulfate and ammonium lauryl sulfate.

The moisturizing agent is selected from the group consisting of ethyleneglycol, propylene glycol, glycerin, sorbitol, low molecular weightpolyethylenes, polyoxyethylated glycerols, polyoxyethylated sugars, forexample, MEA acetamide, and the like. Preferably, the moisturizing agentis glycerin, which has a wide variety of applications, such asemulsifier, softening agent, laminate, stabilizing and moisturizingagent for baking shop, ice-cream shop and cigar store, in body lotions,mouthwashes, as protecting means for freezing of red blood cells, sperm,corneas and other tissues in printing inks, paint resins; antifreezingmixtures; and as raw material for nitroglycerin. It is used in themanufacture of countless prepared pharmaceuticals and cosmetics; forexample in soaps, glycerin increases its detergency, whitening andsoftening of the skin. Between 8-15% of glycerin can be found in thecompositions of these soaps.

Other moisturizing agents useful in the compositions claimed in thepresent invention comprise among others:

A aromatizing agent selected from any of the available ones for thepharmaceutical and food industry.

An aqueous solvent, which is selected from water and water-hydrosolublealcohol mixtures.

With these elements, it is possible to extract a wide range ofantimicrobial, exfoliating and seboregulating pharmaceuticalcompositions containing DDMO and alpha-hydroxyethanoic acid (glycolicacid), useful in treating and preventing skin microbial injuries.

Said compositions contribute to eliminate bacteria causing acneproviding a deep and delicate cleansing of the skin; even in oily skins,improving the habitual look and freshness. In addition to itsantimicrobial action, it provides an exfoliating action, reducing theepidermal adhesion and facilitating the cellular change, eliminatingdead skin cells. Also, they remove the grease and oil excess present inthe acne skin, thus preventing obstruction of the pores, removingcomedonal block and avoiding formation of new blackheads and pustules.

The information obtained from microbiologic tests (antimicrobialchallenge) of the DDMO and alpha-hydroxyethanoic acid (glycolic acid)against several bacteria, has proven the great antibacterial capacity ofthe formulations, insisting on the results against Propionibacteriumacnes, thus proving the removal of said bacteria in a matter of seconds.

The antimicrobial action mechanism of DDMO, probably comprises thefollowing:

1. Alteration of the microbial mechanism, inhibiting the action of 11essential enzymes for cellular respiration of the microorganism. Thisinhibiting effect is created from the chemical reaction of thesulfhydryl radicals of DDMO about the molecular structure of thesulfurated enzymes of the microorganism, whether by inserting sulfuratedradicals or by modifying the enzyme protein, thus causing death of themicroorganism.

2. Through reactions of oxide-reduction creating free radicals (ROS),which by oxidative stress break the cellular wall of the microorganism,by acting “sequestering” oxygen serving as link between the NAG(n-Acetyl glucosamine acid) and NAM (N-Acetyl muramic acid) chains, thusweakening the microorganism wall causing rupture and thereby death ofthe microorganism.

3. Inhibition of the genetic transcription process and unfolding of themicroorganism proteins, through the activation of the polymerase ARNenzyme, which is an enzyme, since being of sulfurated composition, isvulnerable to chemical attack from the DDMO.

4. Alteration of the metal metabolism in the microorganism, also causingmetabolic alterations therein.

The foregoing has been inferred from studies made with allicin, reportedby Yosuda et al, Biosci. Biotechnol. Biochem., 63(3) 591-594, 1999;Feldherg et al, Antimicrobial Agents and Chemotherapy, December 1988,1763-1768; Münchberg et al, Org. Biomol. Chem., 2007, 5, 1505-1518;among others. Considering that DDMO is an stabilized allicin moleculeowing its antimicrobial action precisely to this allicin fragment, whoseactivity is preserved without alteration as shown in the microbiologicaltests.

The microbiological challenges were made with collection microorganisms,universally required to test the product effectiveness. The resultsproved that in 30 seconds of contact with the composition from Example1, 100.00% was removed from the challenged microorganisms, includingCandida albicans. The results from the study are shown in Table 1.

TABLE 1 Studies from microbial challenge Test Microorganism Contact Time30 Sec Escherichia coli 100% ATCC 10536 Pseudomona aerugiosa 100% ATCC9027 Klebsiellapneumoniae 100% ATCC 10031 Proteus vulgaris 100% ATCC6380 Staphylococcus aureus 100% ATCC 6538 Staphylococcus epidermis 100%ATCC 12228 Candida albicans 100% ATCC 1031

Additionally, challenge tests were made against Propionilbacteriumacnes, anaerobic bacteria previously referred to as one of the maincauses of infection caused by acne, thus obtaining excellent results,such as it is shown below in Table 2.

TABLE 2 Contact Time Eradication Test Microorganism 30 seconds 60seconds Propionibacterium acnes 98% 99.999%

The results of these microbial challenges are more encouraging thatthose reported by Fujisawa et al, Biosci. Biotechnol. Biochem. 73 (9)1984-1955, 2009; Tsao et al Journal of Medical Microbiology (2007) 56,803-808. And they confirm the review by Ankri and Mirelman, Microbes andInfection, 2, 1999, 125-129 and by Domingo and Lopez-Brea in Rev. Esp.Quimioterap. December 2003, Vol. 16 (no. 4) 285-293 (2003).

Example 1

An antimicrobial and antiseptic hydrosoluble composition was preparedcontaining:

Component Kg a-hydroxyethanoic acid 57.1 DDMO 4 Sodium lauryl sulfate 50Sodium lauryl ether sulfate 50 Glycerin 2.5 Blue fragrance 3 Purifiedwater 333.4

Example 2

A second composition was prepared containing the following elements:

Component Kg a-hydroxyethanoic acid 58 DDMO 5 Sodium lauryl sulfate 40Sodium lauryl ether sulfate 60 Ethylene glycol 2.5 Floral fragrance 3Purified water 331.5

Example 3

A third example of the object compositions of the present invention isshown, thus proving that the embodiments of the same only limit topreserving the homogeneous distribution aspects of all components.

Component Kg a-hydroxyethanoic acid 57.1 DDMO 4 Sodium lauryl ethersulfate 100 Sorbitol 2 Blue fragrance 3 Purified water-ethanol (9:1)333.9Clinical Trials:

A clinical trial was made applying the product of the present invention(example 1) during 28 days to 20 patients with moderate acne, accordingto the classification shown in Table 3 below.

TABLE 3 Grade* Description 0 Clean skin with no inflammatory ornon-inflammatory wounds 1 Almost clean; Little non-inflammatory woundsand no more tan two inflammatory wounds 2 Middle severity; greater thangrade 1; some non-inflammatory wounds, few inflammatory wounds (onlypapules/pustules; no nodular wounds) 3 Moderate severity; greater thangrade 2; many non-inflammatory wounds and several can be inflammatory,but no more than two nodular wounds. 4 Severe; greater than grade 3;many inflammatory and non- inflammatory wounds; and some nodular wounds.

 FDA's classification scale of acne

The number of wounds from each patient was counted by two differentobservers before treatment and during the same each week. The resultingdata is shown in table 4, wherein it can be seen that the treatment gavean average reduction close to 35% of the active wounds after 28 days oftreatment.

TABLE 4 No. Of Inflammatory Wounds Number Day 0 7 days 14 days 21 days28 days 1 58 +/− 5 51 +/− 5 45 +/− 4 41 +/− 3 37 +/− 3

On the other hand, patients were asked to assess the presence of grease,through the use of drying paper, measuring the number of sheets neededin order not to get the forehead stained 2 hours after cleaning with theproduct, thus discovering that patients considerably reduced the numberof drying sheets used for cleaning the grease, the results are shown inTable 5.

TABLE 5 No. Of Sheets Day 0 7 days 14 days 21 days 28 days 5 +/− 2 2 +/−2 1 +/− 2 1 +/− 1 1 +/− 1

The results show that the antiseptic, antiseborrheic and exfoliatingcomposition of the present invention is able to achieve an effectivetreatment for seborrhea and acne. These results show that the treatmentfor these disorders according to the present invention is clearly betterthan the pre-existing methods.

The invention claimed is:
 1. A hydrosoluble, topical pharmaceutical composition comprising from 0.001 to 10% by weight of diallyl disulfide modified oxide, from 1 to 12% by weight of glycolic acid, from 0.3 to 24% by weight of one or more surfactants, from 0.3 to 10% by weight of a moisturizing agent, from 0.2 to 10% by weight of an aromatizing agent, and from 40 to 70% by weight of an aqueous solvent.
 2. The composition according to claim 1, wherein the surfactants are selected from the group consisting of linear alkylbenzensulfonates and ionic alkyl sulfates.
 3. The composition according to claim 1, wherein the surfactants are selected from the group consisting of sodium lauryl sulfate, sodium lauryl ether sulfate, and ammonium lauryl sulfate.
 4. The composition according to claim 1, wherein the moisturizing agent is selected from the group consisting of ethylene glycol, propylene glycol, glycerin, sorbitol, low molecular weight polyethylenes, polyoxyethylated glycerols, and polyoxyethylated sugars.
 5. The composition according to claim 1, wherein the solvent is selected from water and water-hydrosoluble alcohol mixtures.
 6. A method for removing or reducing acne in a subject in need thereof, the method comprising administering the hydrosoluble topical pharmaceutical composition of claim 1 to skin of the subject.
 7. A topical pharmaceutical composition, comprising: a) diallyl disulfide modified oxide; b) glycolic acid; c) a surfactant selected from the group consisting of (i) sodium lauryl sulfate, (ii) sodium lauryl ether sulfate, and (iii) sodium lauryl sulfate and sodium lauryl ether sulfate; and d) a moisturizing agent selected from the group consisting of glycerin, ethylene glycol, and sorbitol.
 8. The composition of claim 7, wherein the diallyl disulfide modified oxide is 0.8% by weight of the composition.
 9. The composition of claim 7, wherein the diallyl disulfide modified oxide is 1.0% by weight of the composition.
 10. The composition of claim 7, wherein the moisturizing agent is glycerin.
 11. The composition of claim 7, wherein the surfactant is 20% by weight of the composition.
 12. The composition of claim 7, wherein the moisturizing agent is 0.4% by weight of the composition.
 13. The composition of claim 7, wherein the moisturizing agent is 0.5% by weight of the composition.
 14. The composition of claim 7, wherein the glycolic acid is 11.42% by weight or 11.6% by weight of the composition.
 15. The composition of claim 1, wherein the composition kills Propionibacterium acnes.
 16. The composition of claim 7, wherein the composition kills Propionibacterium acnes. 